Abstract
Since its introduction by Fields and Song (1), the two-hybrid system has been widely used to identify and explore interactions between proteins. The extreme popularity of this method has led to numerous modifications. These include many new plasmids and strains for use in the traditional yeast transcription-based assay and the adaptation of the two-hybrid system to other organisms (2–4) and to protein-nucleic acid interactions (one- and three-hybrid systems) (5–7), as well as the development of assays that do not depend on transcription (8,9). These latter modifications are described in detail in Chapters 15–20. This chapter focuses on the many options available for performing a traditional yeast transcription-based two-hybrid experiment.
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References
Fields, S. and Song, O. (1989) A novel genetic system to detect protein:protein interactions. Nature 340, 245.
Dang, C. V., Barret, J., Villa-Garcia, M., Resar, L. M. S., Kato, G. J., and Fearon, E. R. (1991) Intracellular leucine zipper interactions suggest c-myc hetero-oligomerization. Mol. Cell. Biol. 11, 954–962.
Rossi, F., Charlton, C. A., and Blau, H. M. (1997) Monitoring protein-protein interactions in intact eukaryotic cells by b-galactosidase complementation. Proc. Natl. Acad. Sci. USA 94, 8405–8410.
Karimova, G., Pidoux, J., Ullmann, A., and Ladant, D. (1998) A bacterial two-hybrid system based on a reconstituted signal transduction pathway. Proc. Natl. Acad. Sci. USA 95, 5752–5756.
Li, J. J. and Herskowitz, I. (1993) Isolation of ORC6, a component of the yeast origin of recognition complex by a one-hybrid system. Science 262, 1870–1873.
Inouye, C, Remondelli, P., Karim, M., and Elledge, S. (1994) Isolation of a cDNA encoding a metal response element binding protein using a novel expression cloning procedure: the one-hybrid system. DNA Cell Biol. 13, 731–742.
SenGupta, D. J., Zhang, B., Kraemer, B., Pochart, P., Fields, S., and Wickens, M. (1996) A three-hybrid system to detect RNA-protein interactions in vivo. Proc. Natl. Acad. Sci. USA 93, 8496–8501.
Johnsson, N. and Varshavsky, A. (1994) Split ubiquitin as a sensor of protein interactions in vivo. Proc. Natl. Acad. Sci. USA 91, 10,340–10,344.
Aronheim, A., Zandi, E., Hennemann, H., Elledge, S., and Karin, M. (1997) Isolation of an AP-1 repressor by a novel method for detecting protein-protein interactions. Mol. Cell. Biol. 17, 3094–3102.
Chien, C. T., Bartel, P. L., Sternglanz, R., and Fields, S. (1991) The two-hybrid system: a method to identify and clone genes for proteins that interact with a protein of interest. Proc. Natl. Acad. Sci. USA 88, 9578–9582.
Golemis, E. A. and Khazak, V. (1997) Alternative yeast two-hybrid systems: the interaction trap and interaction mating. Methods Mol. Biol. 63, 197–218.
Dagher, M. C. and Filhol-Cochet, O. (1997) Making hybrids of two-hybrid systems. Biotechniques 22, 916–918.
Fromont-Racine, M., Rain, J.-C., and Legrain, P. (1997) Toward a functional analysis of the yeast genome through exhaustive two-hybrid screens. Nat. Genet. 16, 277–282.
Louvet, O., Doignon, F., and Crouzet, M. (1997) Stable DNA-binding yeast vector allowing high-bait expression for use in the two-hybrid system. Biotechniques 23, 816–818.
Hoffman, C. S. and Winston, F. (1987) A ten-minute DNA preparation from yeast efficiently releases autonomous plasmids for transformation of Escherichia coli. Gene 57, 267–272.
Ward, A. C. (1990) Single-step purification of shuttle vectors from yeast for high frequency back-transformation into E. coli. Nucleic Acids Res. 18, 5319.
Chevray, P. M. and Nathans, D. (1992) Protein interaction cloning in yeast: identification of mammalian proteins that react with the leucine zipper of Jun. Proc. Natl. Acad. Sci. USA 89, 5789–5793.
Durfee, T., Draper, O., Zupan, J., Conklin, D. S., and Zambryski, P. C. (1999) New tools for protein linkage mapping and general two hybrid screening. Yeast 15, 1761–1768.
Beranger, F., Aresta, S., de Gunzberg, J., and Camonis, J. (1997) Getting more from the two-hybrid system: N-terminal fusions to LexA are efficient and sensitive baits for two-hybrid studies. Nucleic Acids Res. 25, 2035–2036.
Brown, M. A. and MacGillivray, R. T. A. (1997) Vectors for expressing proteins at the amino-terminus of an activation domain for use in the yeast two hybrid system. Anal. Biochem. 247, 451,452.
Legrain, P., Dokhelar, M.-C., and Transy, C. (1994) Detection of protein-protein interactions using different vectors in the two-hybrid system. Nucleic Acids Res. 22, 3241,3242.
Gietz, R. D., Triggs-Raine, B., Robbins, A., Graham, K. C., and Woods, R. A. (1997) Identification of proteins that interact with a protein of interest: applications of the yeast two-hybrid system. Mol. Cell. Biochem. 172, 67–79.
James, P., Halladay, J., and Craig, E. A. (1996) Genomic libraries and a host strain designed for highly efficient two hybrid selection in yeast. Genetics 144, 1425–1436.
Gyuris, J., Golemis, E. A., Chertkov, H., and Brent, R. (1993) Cdi1, a human G1 and S phase protein phosphatase that associates with Cdk2. Cell 75, 791–803.
Bai, C. and Elledge, S. J. (1996) Gene identification using the yeast two-hybrid system. Methods Enzymol. 273, 331–347.
Fields, S. and Sternglanz, R. (1994) The two-hybrid system: an assay for protein-protein interactions. Trends Genet. 10, 286–292.
Estojak, J., Brent, R., and Golemis, E. A. (1995) Correlation of two-hybrid affinity data with in vitro measurements. Mol. Cell. Biol. 15, 5820–5829.
Brent, R. and Finley, R. L. (1997) Understanding gene and allele function with two hybrid methods. Annu. Rev. Genet. 31, 663–704.
Serebriiskii, I., Khazak, V., and Golemis, E. A. (1999) A two-hybrid dual bait system to discriminate specificity of protein interactions. J. Biol. Chem. 274, 17,080–17,087.
Le Douarin, B., Pierrat, B., vom Baur, E., Chambon, P., and Losson, R. (1995) A new version of the two-hybrid assay for detection of protein-protein interactions. Nucleic Acids Res. 23, 876–878.
Gill, G., and Ptashne, M. (1988) Negative effect of the transcriptional activator GAL4. Nature 334, 721–724.
Bartel, P. L., Chien, C.-T., Sternglanz, R., and Fields, S. (1993) Using the two-hybrid system to detect protein-protein interactions, in Cellular Interactions in Development: A Practical Approach (Hartley, D. A., ed.). Oxford University Press, Oxford, pp. 153–179.
Roder, K. H., Wolf, S. S., and Schweizer, M. (1996) Refinement of vectors for use in the two-hybrid system. Anal. Biochem. 241, 260–262.
Cormack, R. S. and Somssich, I. E. (1997) Dampening of bait proteins in the two-hybrid system. Anal. Biochem. 248, 184–186.
Aho, S., Arffman, A., Pummi, T., and Uitto, J. (1997) A novel reporter gene MEL1 for the yeast two-hybrid system. Anal. Biochem. 253, 270–272.
Uetz, P., Giot, L., Cagney, G., et al. (2000) A comprehensive analysis of protein-protein interactions in Saccharomyces cerevisiae. Nature 403, 623–627.
Hudson, J. R., Dawson, E. P., Rushing, K. L., et al. (1997) The complete set of predicted genes from Saccharomyces cerevisiae in a readily usable form. Genet. Res. 7, 1169–1173.
Durfee, T., Becherer, K., Chen, P. L., et al. (1993) The retinoblastoma protein associates with the protein phosphatase type 1 catalytic subunit. Genes Dev. 7, 555–569.
Harper, J. W., Adami, G., Wei, N., Keyomarsi, K., and Elledge, S. J. (1993) The p21 Cdk-interacting protein Cip1 is a potent inhibitor of G1 cyclin-dependent kinases. Cell 75, 805–816.
Mumberg, D., Muller, R., and Funk, M. (1995) Yeast vectors for the controlled expression of heterologous proteins in different genetic backgrounds. Gene 156, 119–122.
Bartel, P. L. and Fields, S. (1995) Analyzing protein-protein interactions using two-hybrid system. Methods Enzymol. 254, 241–263.
Watson, M. A., Buckholz, R., and Weiner, M. P. (1996) Vectors encoding alternative antibiotic resistance for use in the yeast two-hybrid system. Biotechniques 21, 255–259.
Shaywitz, D. A., Espenshade, P. J., Gimeno, R. E., and Kaiser, C. A. (1997) COPII subunit interactions in the assembly of the vesicle coat. J. Biol. Chem. 272, 25,413–25,416.
Zervos, A. S., Gyuris, J., and Brent, R. (1993) Mxi1, a protein that specifically interacts with Max to bind Myc-Max recognition sites. Cell 72, 223–232.
Vojtek, A. B., Hollenberg, S. M., and Cooper, J. A. (1993) Mammalian Ras interacts directly with the serine/threonine kinase Raf. Cell 74, 205–214.
Dalton, S. and Treisman, R. (1992) Characterization of SAP-1, a protein recruited by serum response factor to the c-fos serum response element. Cell 68, 597–612.
Yavuzer, U. and Goding, C. R. (1995) pWITCH: a versatile two-hybrid assay vector for the production of epitope/activation domain-tagged proteins both in vitro and in yeast. Gene 165, 93–96.
Buckholz, R. G., Simmons, C. A., Stuart, J. M., and Weiner, M. P. (1999) Automation of yeast two hybrid screening. J. Mol. Microb. Biotechnol. 1, 135–140.
Bendixen, C., Gangloff, S., and Rothstein, R. (1994) A yeast mating-selection scheme for detection of protein-protein interactions. Nucleic Acids Res. 22, 1778,1779.
Choi, K. Y., Satterberg, B., Lyons, D. M., and Elion, E. A. (1994) Ste5 tethers multiple protein kinases in the MAP kinase cascade required for mating in S. cerevisiae Cell 78, 499–512.
Marcus, S., Polverino, A., Barr, M., and Wigler, M. (1994) Complexes between STE5 and components of the pheromone-responsive mitogen-activated protein kinase module. Proc. Natl. Acad. Sci. USA 91, 7762–7766.
Printen, J. A. and Sprague, G. F. (1994) Protein-protein interactions in the yeast pheromone response pathway: Ste5p interacts with all members of the MAP kinase cascade. Genetics 138, 609–619.
Keegan, K. and Cooper, J. A. (1996) Use of the two-hybrid system to detect the association of the protein-tyrosine-phosphatase, SHPTP2, with another SH2-containing protein, Grb7. Oncogene 12, 1537–1544.
Leanna, C. A. and Hannink, M. (1996) The reverse two-hybrid system: a genetic scheme for selection against specific protein/protein interactions. Nucleic Acids Res. 24, 3341–3347.
Vidal, M., Brachman, R. K., Fattaey, A., Harlow, E., and Boeke, J. D. (1996) Reverse two-hybrid and one-hybrid systems to detect dissociation or protein-protein and DNA-protein interactions. Proc. Natl. Acad. Sci. USA 93, 10,315–10,320.
Vidal, M., Braun, P., Chen, E., Boeke, J. D., and Harlow, E. (1996) Genetic characterization of a mammalian protein-protein interaction domain by using a yeast reverse two-hybrid system. Proc. Natl. Acad. Sci. USA 93, 10,321–10,326.
Cormack, R. S., Hahlbrock, K., and Somssich, I. (1998) Isolation of putative plant transcriptional coactivators using a modified two-hybrid system incorporating a GFP reporter gene. Plant J. 14, 685–692.
Bartel, P. L., Chien, C.-T., Sternglanz, R., and Fields, S. (1993) Elimination of false positives that arise in using the two-hybrid system. Biotechniques 14, 920–924.
Feilotter, H. E., Hannon, G. J., Ruddell, C. J., and Beach, D. (1994) Construction of an improved host strain for two hybrid screening. Nucleic Acids Res. 22, 1502, 1503.
Ausebel, F. R., Brent, R., Kingston, R., et al. (1989) Current Protocols in Molecular Biology, John Wiley & Sons, New York.
Agatep, R., Kirkpatrick, R. D., Parchaliuk, D. L., Woods, R. A., and Gietz, R. D. (1998) Transformation of Saccharomyces cerevisiae by the lithium acetate/single-stranded carrier DNA/polyethylene glycol (LiAc/ss-DNA/PEG) protocol. Technical Tips Online (http://tto.trends.com).
Rose, M. D., Winston, F., and Hieter, P. (1990) Methods in Yeast Genetics: A Laboratory Course Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
El Housni, H., Vandenbroere, I., Perez-Morga, D., Christophe, D., and Pirson, I. (1998) A rare case of false positive in a yeast two-hybrid screening: the selection of rearranged bait constructs that produce a functional Gal4 activity. Anal. Biochem. 262, 94–96.
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James, P. (2001). Yeast Two-Hybrid Vectors and Strains. In: MacDonald, P.N. (eds) Two-Hybrid Systems. Methods in Molecular Biology, vol 177. Humana Press. https://doi.org/10.1385/1-59259-210-4:041
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DOI: https://doi.org/10.1385/1-59259-210-4:041
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